Particle-mediated delivery of biologics

ABSTRACT

A composition for delivering a biologic to a subject generally includes a particulate substrate and an mRNA encapsulated by the particulate substrate. In some cases, the mRNA bay be indirectly attached to the particulate substrate. The mRNA encodes at least one therapeutic polypeptide. The composition may be delivered to a tissue of a subject to provide a therapeutic benefit to the tissue.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/426,090, filed Nov. 23, 2016, which is incorporated herein byreference in its entirety.

BACKGROUND

A heart attack, also called a myocardial infarction, occurs when a partof the heart muscle does not receive enough blood flow. The more timethat passes without treatment to restore blood flow, the greater thedamage to the heart muscle. Every year, about 735,000 Americans have aheart attack, including about 210,000 heart attacks that happen topeople who have already had a first heart attack (Mozaffarian et al.,Circulation, 131(4):e29-322 (2015)).

SUMMARY

This disclosure describes modifications to RNA molecules that increasethe length of time that the modified RNA molecules exist in cells.

This disclosure further provides materials and methods forparticle-mediated delivery of one or more biologics (e.g., RNA, modifiedRNA, and/or microvesicles) to a tissue (e.g., heart tissue).Microvesicles can represent manufactured particles or naturallyoccurring structures, such as exosomes. For example, this documentprovides particles (e.g., alginate gels) for delivering one or more RNAsto cardiac tissue to improve cardiac function.

As demonstrated herein, an alginate gel can be used to deliver mRNA to aheart tissue where the mRNA can be translated into a functionalpolypeptide. Particle-mediated delivery of RNA can induce robust andsustainable RNA expression. In some cases, particles can be used bedesigned to control temporal and/or spatial delivery of one or moreencapsulated molecules (e.g., biologics).

In one aspect, therefore, this disclosure describes composition thatgenerally include a particulate substrate and an mRNA attached to theparticulate substrate. The mRNA includes at least one modification toinhibit degradation of the mRNA when the mRNA is in cytosol of a cell.The mRNA also encodes at least one therapeutic polypeptide. In someembodiments, the mRNA modification comprises a pseudoknot, an RNAstability element, or an artificial 3′ stem loop. In some embodiments,the particulate substrate can include a chemical modification of itssurface. In various embodiments, the particulate substrate can include ananoparticle, a plurality of nanoparticles, or a microparticle. In someembodiments, the therapeutic polypeptide can include an immunoglobulinheavy chain or an immunoglobulin light chain.

In general, one aspect of this document features a method for improvingcardiac function. The method includes, or consists essentially of,administering a particle encapsulating an mRNA encoding a polypeptideuseful for regenerating cardiac function and/or tissue to a mammal,thereby improving cardiac function of the mammal. The polypeptide can beNAP-2, TGF-a, ErBb3, VEGF, IGF-1, FGF-2, PDGF, IL-2, CD19, CD20, and/orCD80/86. The mammal can be a human. The human can have undergonepercutaneous coronary intervention for ST-elevation myocardialinfarction. The administering can be an arterial administration. Theparticle can include alginate. The alginate can be in the form of analginate gel. The alginate gel can include a calcium salt. The alginategel including a calcium salt can have a ratio of alginate to calciumsalt that can be from about 2:1 to about 10:1. The particle can be fromabout 5 μm to about 10 μm in diameter. The particle can be a biphasicparticle. The biphasic particle can be a polarized particle. Thebiphasic particle can have a tail. The method can include administeringthe composition during a percutaneous coronary intervention. Theparticle can include a scaffold protein (e.g., collagen I, collagen II,collagen III, collagen IV, fibrin, and/or gelatin). The particle canencapsulate a polypeptide (e.g., an antibody having the ability toneutralize tumor necrosis factor activity, an antibody having theability to neutralize mitochondrial complex-1 activity, or a resolvin-D1agonist). The particle can encapsulate a lipopolysaccharide. Theparticle can encapsulate a microvesicle and/or exosome.

In another aspect, this document features a method for improving cardiacfunction in a mammal. The method includes, or consists essentially of,administering to a mammal a particle encapsulating an inhibitory RNAhaving the ability to reduce expression of a polypeptide selected fromthe group consisting of eotaxin-3, cathepsin-S, DK-1, follistatin, ST-2,GRO-a, IL-21, NOV, transferrin, TIMP-2, TNFaRI, TNFaRII, angiostatin,CCL25, ANGPTL4, and MMP-3, thereby improving cardiac function of themammal. The mammal can be a human. The human can have undergonepercutaneous coronary intervention for ST-elevation myocardialinfarction. The administering can be an arterial administration. Theparticle can include alginate. The alginate can be in the form of analginate gel. The alginate gel can include a calcium salt. The alginategel including a calcium salt can have a ratio of alginate to calciumsalt that can be from about 2:1 to about 10:1. The particle can be fromabout 5 μm to about 10 μm in diameter. The particle can be a biphasicparticle. The biphasic particle can be a polarized particle. Thebiphasic particle can include a tail. The method can includeadministering the composition during a percutaneous coronaryintervention. The particle can include a scaffold protein (e.g.,collagen I, collagen II, collagen III, collagen IV, fibrin, and/orgelatin). The particle can encapsulate a polypeptide (e.g., an antibodyhaving the ability to neutralize tumor necrosis factor activity, anantibody having the ability to neutralize mitochondrial complex-1activity, or a resolvin-D1 agonist). The particle can encapsulate alipopolysaccharide. The particle can encapsulate a microvesicle and/orexosome.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematics illustrations of exemplary methods for making biphasicparticles. (A) A stabilizer technology method where two separate softliquid cores, such as a water-based component and an oil-basedcomponent, are stabilized and then merged into a single Janus particle.(B) A melt combination and fusion method where two polymer streams aremerged together forming a single particle and cooled to form twohemispheres of a Janus particle. (C) A microfluidics method that usescontrolled liquid flow in microfluidic channels to form droplets thatare solidified into formed Janus particles.

FIG. 2. Schematic illustrations of exemplary delivery methods usingbiphasic particles. (A) A Janus particle with different volume percentor density could orient the particle within the blood flow and directthe biologics in an appropriate orientation for delivery. (B) A Janusparticle having a coating can control the rate of release and/or augmentorienting capability. (C) A Janus particle having a tail (top), havingdifferential solubility (middle), and/or having differential positioningof microcapsules on either end of the Janus particle (bottom) canprovide orienting guidance to the particle.

FIG. 3. Fluorescent (first and third rows) and light phase (second andfourth rows) microscopy images showing mCherry transfection in humandermal fibroblasts (top two rows) and human cardiac fibroblasts (bottomtwo rows).

FIG. 4. Fluorescent microscopy images showing co-transfection of EGFPand mCherry in HEK 293 cells.

FIG. 5. Fluorescent (top row) and light phase (bottom row) microscopyimages showing transfection of mCherry in HL-1 cardiomyocytes.

FIG. 6. Data showing particle-mediated delivery of mRNA in a mousemodel. (A) Tail vein hydrodynamic injection of a control solution (CTL).(B) Tail vein hydrodynamic injection of liposomes containing luciferasemRNA. (C) Subcutaneous injection of a control solution (CTL). (D)Subcutaneous injection of luciferase mRNA. (E) A bar graph showing theamount of luciferase expressed by the mice in (A) and (B). (F) A bargraph showing the amount of luciferase expressed by the mice in (C) and(D).

FIG. 7. Microscopy images showing mCherry expression in mice subjectedto subcutaneous injection of a control solution (vehicle only; top row)or liposomes containing mCherry RNA (bottom row).

FIG. 8. Data showing particle-mediated delivery of mRNA in a mousemodel. (A) A photograph of mice injected with a control solution orliposomes containing luciferase mRNA by echo guided intracardiacinjection. (B) A bar graph showing the amount of luciferase expressed bythe mice in (A).

FIG. 9. Photographs of mouse hearts injected with a control solution orliposomes containing luciferase mRNA by open chest intracardiacinjection.

FIG. 10. A photograph of a cross-section of a porcine heart injectedwith an alginate gel m-Cherry reporter system.

FIG. 11. Fluorescent images of porcine hearts injected with a reducedalginate gel volume.

FIG. 12. Fluorescent images of porcine hearts injected with variedalginate/calcium concentrations.

FIG. 13. Fluorescent images following administration of alginate spherescontaining mCherry mRNA.

FIG. 14. A needle design for efficient biologics delivery into tissues,featuring a micro-spiral design and side holes.

FIG. 15. Several RNA designs modifying the 5′CAP and the poly(A) tailfor sustained gene expression in vivo without DNA integration. (A)Native mRNA. (B) Loop engineered modified mRNA adding a protective loopto the end of the poly(A) tail. (C) Loop poly(A) tail plus modificationof the 5′ 7-methyl guanosine cap to further limit degradation.

FIG. 16. Transfection of HEK cells will M²RNA encoding mCherry resultsin rapid gene induction that is sustained for a six-day observationperiod.

FIG. 17. Data showing persistent expression of M³RNA. (A) M³RNA inducesmCherry in primary cultured cardiomyocytes. Temporal visualization ofmCherry expression over a six-day observation period indicatespersistence of quantified expression. (C) Flow cytometric evaluationreveals 43% efficiency in inducing cultured cardiomyocytes.

FIG. 18. Primary human skeletal muscle culture system. Derived fromdonor samples, master cell bank of P3 myoblasts are generated within adefined xeno-free propagation medium. Within a differentiationenvironment MyoD⁺ cells can be induced to differentiate intoactinin-positive myotubes.

FIG. 19. M³RNA-mediated dual gene expression within a singleencapsulation system. Induction of both GFP and mCherry within fourhours and persistent expression over a 24-hour observation period withinprimary cultures skeletal muscle cells.

FIG. 20. In vivo delivery of Fluc M³RNA demonstrates compatibility ofthis platform in a broad array of tissues. Sham controls are providedfor each example with the exception of the eye, where the contralateraleye served as sham control.

FIG. 21. Quantification of Fluc expression following delivery of M³RNAvia direct myocardial injection over a 72-hour observation period.

FIG. 22. Evidence for simultaneous delivery of three genes using theM³RNA platform with direct myocardial injection.

FIG. 23. Vector design strategy for establishment of the M³RNA-Igplatform.

FIG. 24. Use of IRES sequence eliminates the need for a CAP/PABPdependent system.

FIG. 25. 3′ strategies to diminish the rate of mRNA degradation focuseson 3 putative platforms. The Pseudoknot mediates ribosomal read-throughknocking off UPF1 molecules.

The RNA stability element acts as a decoy to block UPF1 contact with the3′UTR avoiding activation of the NMD. Poly(A) tail stem loop structuresare used to diminish exosome-mediated mRNA degradation in constructswhere a CAP/PABP independent IRES platform is used.

FIG. 26. Combination of M²RNA with microparticles coated with PEG andchitosan yields the putative M³RNA-Ig platform optimized for genedelivery in skeletal muscle.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

This disclosure relates to materials and methods for particle-mediateddelivery of biologics (e.g., RNA and/or microvesicles) to tissue (e.g.,heart tissue). This disclosure further describes modifications to RNAthat increase the length of time that the modified mRNA molecules existin cells and, therefore, the length of time that the modified mRNAmolecules can be translated into produce polypeptides encoded by themodified mRNA.

This disclosure provides materials and methods for particle-mediateddelivery of one or more biologics (e.g., RNA and/or microvesicles) to amammal. For example, a particle (e.g., an alginate gel) described hereincan be used to encapsulate RNA and deliver the encapsulated RNA to atissue in a temporally and/or spatially specific manner. As used herein,the term “encapsulate” and variations thereof is used broadly to referto any method by which a biologic is housed within a particle or bound,directly or indirectly, to the outer surface of a particle. As explainedin more detail below, the particle can be nanoparticulate ormicroparticulate in scale. Thus, the term “encapsulate” includes, butdoes not require, that the particle completely surround the materialbeing encapsulated. Rather, a material is encapsulated if it is merelycaptured to any degree within the dimensions of the particle, includingany surface modifications. Surface modifications of the particle canfacilitate indirect binding of the encapsulated material to theparticle. In addition, macroencapsulation can be used to deliver abiologic by providing a protective vehicle by which to transmit thebiologics either by direct injection or through a blood vessel into atarget tissue.

In some cases, one or more particles (e.g., alginate gels) containingone or more biologics (e.g., RNAs and/or microvesicles) can be used totreat a mammal experiencing a major adverse cardiac event (e.g., acutemyocardial infarction) and/or a mammal at risk of experiencing a majoradverse cardiac event (e.g., patients who underwent percutaneous intraintervention (PCI) for ST-elevation myocardial infarction (STEMI)).

In some cases, one or more particles (e.g., an alginate gels) containingone or more biologics (e.g., RNAs or microvesicles) can be used toimprove cardiac function.

In some cases, a particle (e.g., an alginate gel) described herein canstore mRNA within its interior, and release (e.g., deliver) the mRNA toa tissue (e.g., heart tissue) where the mRNA is expressed as afunctional protein. For example, one or more particles containing one ormore mRNAs can be used to increase expression of polypeptides useful forregenerating cardiac function and/or tissue (e.g., POU homeodomainproteins (such as Oct-4), NK2 homeobox proteins (e.g., NKX2 proteins),myocyte enhancing factors (e.g., MEF2), GATA binding proteins (e.g.,GATA1, GATA2, GATA3, GATA4, GATA5, and GATA6), T-box transcriptionfactors (e.g., TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18,TBX19, TBX20, TBX21, and TBX22), mesoderm posterior proteins (e.g.,MESP1 and MESP2), neutrophil-activating proteins (e.g., NAP-2 andNAP-3), transforming growth factors (e.g., TGF-α and TGF-β),erythroblastic leukemia viral oncogene-3 (ErBb3), vascular endothelialgrowth factor (VEGF), insulin-like growth factor 1 (IGF-1), fibroblastgrowth factor (FGF-2), platelet-derived growth factors (e.g., PDGFA,PDGFB, PDGFC, and PDGFD), Interleukin-2 (IL-2), CD19, CD20, andCD80/86).

In some cases, a particle (e.g., an alginate gel) described herein canstore inhibitory RNA within its interior, and release (e.g., deliver)the inhibitory RNA to a tissue (e.g., heart tissue) where the inhibitoryRNA inhibits or reduces expression of a protein. For example, one ormore particles containing one or more inhibitory RNAs can be used todecrease expression of one or more of the following polypeptides:eotaxin-3, cathepsin-S, Dickopf-1 (DK-1), follistatin, suppression oftumorigenicity-2 (ST-2), GRO-α, interleukin-21 (IL-21), nephroblastomaoverexpressed (NOV), transferrin, tissue inhibitor of metallopeptidase-2(TIMP-2), tumor necrosis factor receptor-1 and -2 (TNFαRI and II),angiostatin, chemokine ligand-25 (CCL25), angiopoietin like-4 (ANGPTL4),and matrix metalloproteinase-3 (MMP-3).

Particles

A particle described herein can be used for particle-mediated deliveryof one or more molecules (e.g., biologics including RNA ormicrovesicles) to a mammal. In some cases, a particle that can be usedto encapsulate one or more biologics is non-toxic, biocompatible,non-immunogenic, and/or biodegradable.

A particle that can be used to encapsulate one or more biologics asdescribed herein can include one or more polysaccharides. Examples ofpolysaccharides that can be used in a particle that can be used toencapsulate one or more molecules (e.g., biologics) as described hereininclude, for example, guluronate, mannuronate, guluronate-mannuronateblocks, and combinations thereof such as alginate. In some cases, aparticle that can be used to encapsulate one or more biologics asdescribed herein can include alginate. An alginate in a particle thatcan be used to encapsulate one or more biologics as described herein canbe from any appropriate source (e.g., seaweeds such as those in thegenera Phaeophyceae, Rhodophyceae, Chlorophyceae, Macrocystis, andLaminaria, or bacteria such as those in the genera Pseudomonas andAzotobacter). An alginate in a particle that can be used to encapsulateone or more biologics as described herein can be an alginate salt (e.g.,sodium alginate, potassium alginate, and/or calcium alginate) or alginicacid. For example, an alginate in a particle that can be used toencapsulate one or more biologics as described herein can be sodiumalginate. An alginate in a particle that can be used to encapsulate oneor more biologics as described herein can be in the form of a gel, aliquid, and/or a particle (e.g., a nanoparticle, a microparticle, or amacroparticle). For example, an alginate in a particle that can be usedto encapsulate one or more biologics as described herein can be analginate gel. In some cases, a particle described herein can be a liquidat the time of administration and can form a gel (e.g., can polymerize)at the administration site and/or the target site. An alginate gel caninclude a calcium solution (e.g., a calcium salt solution or anotherappropriate positively charged ionic solution). Examples of calciumsalts that can be used in a calcium solution to form an alginate gelthat can be used to encapsulate one or more biologics as describedherein include, without limitation, calcium chloride, magnesiumchloride, potassium chloride, and iron-based solutions. The ratio ofalginate to calcium salt can be used to control the viscosity of thealginate gel that can be used to encapsulate one or more biologics asdescribed herein. In some cases, the amount of alginate can be fromabout 0.05 percent to about 1.0 percent of the amount of calcium salt.In some cases, the ratio of alginate to calcium salt can be can be fromabout 2:1 to about 10:1 (e.g., from about 2:1 to about 9:1, from about2:1 to about 8:1, from about 2:1 to about 7:1, from about 2:1 to about6:1, from about 2:1 to about 5:1, from about 3:1 to about 10:1, fromabout 4:1 to about 10:1, from about 5:1 to about 10:1, from about 6:1 toabout 10:1, from about 7:1 to about 10:1, or from about 8:1 to about10:1). Examples of appropriate alginate/calcium ratios include, withoutlimitation, those set forth in Table 1. An alginate gel encapsulating abiologic (e.g., mRNA) described herein can be made by any appropriatemethod. For example, an alginate gel can be made by hydrolyzing analginate salt (e.g., sodium alginate) using a soluble calcium saltsolution (e.g., calcium chloride) as a crosslinking agent.

TABLE 1 Examples alginate/calcium concentrations. alginate concentrationcalcium concentration alginate:calcium 0.2%   0.1% 2:1 1% 0.1% 10:1  2%0.5% 4:1 2% 0.25%  8:1

A particle that can be used to encapsulate one or more biologics asdescribed herein can be in the form of a liposome, an aggregate (e.g., anano-aggregate), a capsule (e.g., a nanocapsule), a sphere (e.g., ananosphere), a polymersome, or a micelle. A particle that can be used toencapsulate one or more biologics as described herein that is analginate gel can be in the form of polymerized sphere. In some cases, aparticle that can be used to encapsulate one or more biologics asdescribed herein can be a liposome. Examples of liposomes include,without limitation, a multilamellar vesicle (MLV), a small unilamellarliposome vesicle (SUV), a large unilamellar vesicle (LUV), a giantunilamellar vesicle (GUV), a multivesicular vesicles (MVV), or acochleate vesicle. A liposome can be composed of phospholipids,cholesterols, lipoproteins, fats, fatty acids, waxes, sterols,monoglycerides, diglycerides, and/or triglycerides. In some cases, aliposome is composed of phospholipids such as phosphatidic acid(phosphatidate; PA), phosphatidylethanolamine (cephalin; PE),phosphatidylcholine (lecithin; PC), phosphatidylserine (PS),phosphoinositides (e.g., phosphatidylinositol (PI), phosphatidylinositolphosphate (PIP), phosphatidylinositol bisphosphate (PIP2), andphosphatidylinositol triphosphate (PIPS)), ceramide phosphorylcholine(sphingomyelin; SPH), ceramide phosphorylethanolamine (sphingomyelin;Cer-PE), ceramide phosphoryllipid, or any combination thereof. Examplesof appropriate liposomes include, without limitation, those set forth inTable 2. A liposome encapsulating a biologic (e.g., mRNA) describedherein can be made by any appropriate method. For example, a liposomecan be made by sonicating a dispersion of amphipatic lipids, such asphospholipids, in water.

TABLE 2 Examples of liposome components. MW liposome component (kDa)Concentration Charged Poly(lactide-co-glycolide) (PLGA) about 46 3-300mg 1,2-Dioleoyl-sn-glycero-3- about 744 20 μg-1 mg phosphoethanolamine(DOPE) N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- about 774 20 μg-3 mgtrimethylammonium methylsulfate (DOTAP) L-α-Phosphatidylethanolamine,dioleoyl about 744 20 μg-3 mg 1,2-Distearoyl-sn-glycero-3- about 770 20μg-3 mg phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG)Poly(vinyl-alcohol) (PVA) about 78 1-5% per total volume

A particle that can be used to encapsulate one or more biologics asdescribed herein can be any size suitable for the selected deliverymethod. A particle that can be used to encapsulate one or more biologicsas described herein can be from about 0.3 μm to about 12 μm (e.g., fromabout 0.5 μm to about 11.5 μm, from about 1 μm to about 11 μm, fromabout 2 μm to about 10.5 μm, or from about 4 μm to about 10 μm) indiameter (or as measure across the longest dimension). For example, aparticle that can be used to encapsulate one or more biologics asdescribed herein can be from about 4.5 μm to about 7.5 μm in diameter(or as measure across the longest dimension).

A particle (e.g., an alginate gel) encapsulating one or more biologicsdescribed herein also can include one or more additional molecules inorder to achieve a desired property. In some cases, a particleencapsulating one or more biologics described herein can include one ormore scaffold proteins. Examples of scaffold proteins include, forexample, matrix proteins (e.g., collagen (e.g., collagen I/II/III/IV)),basement membrane proteins, structural proteins, gelatin, and/or fibrin)can be incorporated into a particle to provide the particle withsustained release properties. For example, an alginate gel encapsulatingone or more biologics described herein can include alginate and gelatin,alginate and collagen, alginate and fibrin, or any other appropriatecombination of alginate with one or more natural basement membraneproteins. In some cases, stimuli-sensitive molecules can be incorporatedinto a particle to provide the particle with drug release propertiesunder specific stimuli (e.g., pH-sensitive particles andosmolarity/osmolality sensitive particles). For example,dioleoylphosphatidylethanolamine (DOPE) can be incorporated into aparticle to provide a pH-sensitive particle that maintains stability atphysiological pH (about pH 7.4), but destabilize under acidic conditions(e.g., about pH 3.5 to about pH 9) leading to the release of theencapsulated RNA in acidic environments.

A particle (e.g., an alginate gel) encapsulating one or more biologicsdescribed herein can be an amorphous particle having two or moredistinct physical states. For example, an amorphous particle can beadministered as a liquid and form a gel upon arriving at a targettissue.

A particle (e.g., an alginate gel) encapsulating one or more biologicsdescribed herein can be a biphasic particle (sometimes referred to as aJanus particle) having two or more distinct physical properties (e.g.,surface chemistry) occurring on different portions of the particlesurface. For example, a biphasic particle can have two or more portionsthat differ in hydrophilic/hydrophobic properties, polarization,solubility properties, volume percent or density of the particlecomposition, the presence/absence of an additional molecule (e.g., acompound providing sustained release properties), and/or thepresence/absence of a tail. In some cases, a biphasic particle can be aspherical particle having hydrophilic portions and hydrophobic portions.For example, a biphasic particle can be designed to have a hydrophiliccore and a hydrophilic coating. For example, a biphasic particle can bedesigned to have a hydrophilic core and a hydrophilic coating. In somecases, a biphasic particle can be a spherical particle havingdifferential density. For example, a biphasic particle can be designedto have increased volume percent of an encapsulated biologic at one endof the particle. In some cases, a biphasic particle can be a sphericalparticle having a coating. For example, a biphasic particle can bedesigned to have a porous coating. In some cases, a biphasic particlecan be a spherical particle having differential solubility. For example,a biphasic particle can be designed to have a first side that readilydissolves in certain physiological conditions and a second side thatdissolves slowly in the same physiological conditions. In some cases, abiphasic particle can be a spherical particle having a tail extendingfrom one portion of the particle surface. For example, a biphasicparticle can be designed to guide particles in the blood flow. In somecases, a biphasic particle can be designed to have a first (e.g.,leading) side that readily dissolves in certain physiological conditionsand a second side that dissolves slowly in the same physiologicalconditions and includes a tail to guide particles in the blood flow.

A biphasic particle encapsulating one or more biologics described hereincan be made using any appropriate method. In some cases, a biphasicparticle can be made using stabilizer technology (e.g., including drywater approaches). For example, two or more separate soft liquid cores(e.g., two liquids having different hydrophobicity such as a hydrophobicentity and hydrophilic entity, or two liquids having differentsolubility such as water and an oil) can be stabilized and then mergedinto a biphasic particle. In some cases, a biphasic particle can be madeusing melt combination and fusion technology. For example, two or moreseparate streams (e.g., two streams of molten wax having differentialproperties, or two streams of polymers having different properties) canbe merged (e.g., injected into a single stream) to form a biphasicparticle. In some cases, a biphasic particle can be made using3D-printer technology. For example, two or more coatings (e.g., two waxcoatings having different properties) can be printed onto a particulateinternal structural element (e.g., a particle of polystyrene foam suchas STYROFOAM′) to form a biphasic particle. The particulate internalstructural element can be removed (e.g., by acetone elimination) andreplaced with one or more biologics. In some cases, a biphasic particlecan be made using microfluidics technology. For example, two or moreliquid flows (e.g., two liquids having different properties) can beflowed into a microfluidic channel to form biphasic droplets, which canbe solidified (e.g., by thermal polymerization, elimination of sheerstress on spatially divalent particles, evaporation, coagulation, cationexposure, or pH) into biphasic particles. In some cases, a biphasicparticle can be made using protection and de-protection technology. Forexample, a protection particle having a desired surface chemistry can beadsorbed onto a main particle; de-protection can yield the originalsurface of the main particle, which can be chemically modified.

In some cases, a biphasic particle can be made use other appropriatetechnologies. For example, other appropriate methods can include,without limitation, interfacial emulsification, acorn and dumbbellshaped formation, use of magnetic fields to shape and manipulateparticles during fabrication, and direct surface coating deposition. Insome embodiments, differential density can be achieved using organogelswith varied density. In some embodiments, differential porosity can beachieved using salts at a particular surface of the particle. In someembodiments, differential solubility can be achieved using salts and/orusing two or more different polymers (e.g., PEGs) having differentsolubilities. In some embodiments, various tails can be achieved usingwaxy components, chemically modifying (e.g., with long-chain reactants),and/or magnetizing a particle. Exemplary methods for making a biphasicparticle encapsulating one or more biologics described herein are shownin FIG. 1.

A particle (e.g. an alginate gel) encapsulating one or more biologicsdescribed herein can be used to deliver the encapsulated biologics to atarget tissue (e.g., heart tissue). Mechanisms of targeting a particledescribed herein can include using an adhesive particle, a targetingmoiety, particle size, capillary leakage in the setting of injury,capillary leakage in the setting of oncogenic neovasculogenesis, and/ordirect injection (e.g., via an edema forming needle (e.g., a nitinol(nickel-titanium) helical needle) designed with graded side holes and noend hole (see, e.g., FIG. 14)). Capillary leakage refers to anyphysical, chemical, or pharmacological means by which one can enhancecapillary porosity or leakage in order to augment biologics delivery.This can be achieved either by administering a factor (e.g., chemicaland/or pharmacologic) that enhances capillary porosity. Alternatively,capillary leakage may be achieved by placing a biologics delivery needlethat is designed to mimic edema into the target tissue.

In some cases, an adhesive moiety can be conjugated to a particledescribed herein to retain an adhesive particle at the administrationsite. For example, an adhesive particle described herein can beadministered (e.g., injected) directly to a target tissue (e.g., cardiacinfarct bed). Examples of adhesive moieties include, without limitation,PEG, positive charge, and self-assembly within tissue. In some cases, atargeting moiety can be conjugated to a particle described herein todirect delivery of a particle to a target tissue (e.g., cardiac infarctbed). Examples of targeting moieties include, without limitation,antigens, tissue targeting peptides, small molecules, and cell surfacemolecules. For example, an antibody can be used to target surfaceproteins on a cell. In some cases, the size of the particle can be usedto direct delivery of a particle to a target tissue (e.g., cardiacinfarct bed). Human capillaries measure about 5 μm to 10 μm in diameter.Thus, a particle described herein having a diameter of from about 0.3 μmto about 12 μm can enter a capillary via the bloodstream, but be limitedfrom exiting the capillary, where the biologics and/or an expressedpolypeptide can diffuse into the capillary bed of a tissue (e.g., heart,dermal, lung, solid tumor, brain, bone, ligament, connective tissuestructures, kidney, liver, subcutaneous, and vascular tissue). Exemplarymethods for delivery of one or more biologics using a biphasic particledescribed herein are shown in FIG. 2.

A particle (e.g., an alginate gel) encapsulating one or more biologicsdescribed herein can be used to deliver the encapsulated biologic to atarget tissue (e.g., cardiac infarct bed) in a temporally and/orspatially specific manner. For example, a biphasic particleencapsulating one or more biologics as described herein can be designedto confer a specific orientation of the particle within a blood vessel,to direct the biphasic particle into a capillary, and/or to conferspecific release conditions of the encapsulated biologics. In caseswhere a biphasic particle has hydrophilic portions and hydrophobicportions on the particle surface, delivery of one or more encapsulatedbiologics can be controlled by the solvent present at the delivery siteand/or the target site. In cases where a biphasic particle hasdifferential density, the differential density can orient the particlein the blood flow to direct the particle in an appropriate orientationfor delivery of one or more encapsulated biologics. For example, a denseside of a biphasic particle can lead the particle in the direction ofblood flow within a blood vessel. In cases where a biphasic particle hasa porous coating, delivery of one or more encapsulated biologics can becontrolled by the differential porosity. For example, different amountsof porosity can help deliver particles within the blood vessel and alterthe pattern of delivery. In cases where a biphasic particle hasdifferential solubility, the differential solubility can orient theparticle in the blood flow to direct the particle in an appropriateorientation for delivery of one or more encapsulated biologics. In caseswhere a biphasic particle has a tail extending from one portion of theparticle surface, the tail can orient the particle within a blood vesselto direct the particle into a capillary. For example, a biphasicparticle can be designed to have a tail to orient the particle within ablood vessel and direct the particle into a blood vessel (e.g., acapillary) where the encapsulated biologic can be delivered.

Biologics

Any appropriate biologic can be encapsulated within a particle describedherein for delivery to a tissue. Examples of biologics that can beencapsulated within a particle described herein include, withoutlimitation, nucleotides, polypeptides, small molecules, microvesicles,exosomes, extracellular vesicles, engineered cells, or combinationsthereof. In some cases, a biologic can be a purified biologic (e.g.,purified microvesicles or exosomes).

A particle (e.g., an alginate gel) described herein can be used toencapsulate one or more polypeptides. In some cases, a particledescribed herein can be used to encapsulate one or more polypeptidesuseful to treat a mammal experiencing a major adverse cardiac event(e.g., acute myocardial infarction) and/or a mammal at risk ofexperiencing a major adverse cardiac event (e.g., patients who underwentPCI for STEMI). For example, a particle encapsulating a biologicdescribed herein can encapsulate one or more polypeptides useful forregenerating cardiac function and/or tissue or a nucleotide that encodessuch a polypeptide. Examples of polypeptides useful for regeneratingcardiac function and/or tissue include, without limitation, antibodieshaving the ability to neutralize tumor necrosis factor (TNF; e.g.,TNF-α) activity, antibodies having the ability to neutralizemitochondrial complex-1 activity, and resolvin-D1 agonists.

A particle (e.g., an alginate gel) described herein can be used toencapsulate one or more nucleotides. Examples of nucleotides that can beencapsulated within a particle include, without limitation, mRNAs,inhibitory RNAs (e.g., antisense RNAs, microRNAs (miRNAs), smallinterfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and agomiRs),antagomiRs, modified mRNAs, loop-engineered modified mRNAs (see, e.g.,FIG. 15), or combinations thereof.

In some cases, a particle (e.g., an alginate gel) described herein canbe used to encapsulate one or more mRNAs useful to treat a mammalexperiencing a major adverse cardiac event (e.g., acute myocardialinfarction) and/or a mammal at risk of experiencing a major adversecardiac event (e.g., patients who underwent PCI for STEMI). For example,a particle encapsulating a biologic described herein can encapsulate oneor more mRNAs encoding a polypeptide useful for regenerating cardiacfunction and/or tissue can be encapsulated within a particle describedherein. Examples of polypeptides that can be useful for regeneratingcardiac function and/or tissue include, without limitation, TNF-α,mitochondrial complex-1, resolvin-D1, NAP-2, TGF-α, ErBb3, VEGF, IGF-1,FGF-2, PDGF, IL-2, CD19, CD20, CD80/86, polypeptides described in WO2015/034897, or an antibody directed against any of the foregoingpolypeptides. For example, a human Nap-2 polypeptide can have the aminoacid sequence set forth in, for example, National Center forBiotechnology Information (NCBI) Accession No. NP_002695.1 (GI No. 5473)and can be encoded by the nucleic acid sequence set forth in NCBIAccession No. NM_002704 (GI No. 5473). In some cases, a human TGF-αpolypeptide can have the amino acid sequence set forth in NCBI AccessionNo. NP_003227.1 (GI No. 7039) and can be encoded by the nucleic acidsequence set forth in NCBI Accession No. NM_003236 (GI No. 7039). Insome cases, a human ErBb3 polypeptide can have the amino acid sequenceset forth in NCBI Accession No. NP_001005915.1 or NP_001973.2 (GI No.2065) and can be encoded by the nucleic acid sequence set forth in NCBIAccession No. NM_001005915.1 or NM_001982.3 (GI No. 2065). For example,a human VEGF can have the amino acids set forth in NCBI Accession Nos.AAA35789.1 (GI: 181971), CAA44447.1 (GI: 37659), AAA36804.1 (GI:340215), or AAK95847.1 (GI: 15422109), and can be encoded by the nucleicacid sequence set forth in NCBI Accession No. AH001553.1 (GI: 340214).For example, a human IGF-1 can have the amino acid sequence set forth inNCBI Accession No. CAA01954.1 (GI: 1247519) and can be encoded by thenucleic acid sequence set forth in NCBI Accession No. A29117.1 (GI:1247518). For example, a human FGF-2 can have the amino acid sequenceset forth in NCBI Accession No. NP_001997.5 (GI: 153285461) and can beencoded by the nucleic acid sequence set forth in NCBI Accession No.NM_002006.4 (GI: 153285460). For example, a human PDGF can have theamino acid sequence set forth in NCBI Accession No. AAA60552.1 (GI:338209) and can be encoded by the nucleic acid sequence set forth inNCBI Accession No. AH002986.1 (GI: 338208). For example, a human IL-2can have the amino acid sequence set forth in NCBI Accession No.AAB46883.1 (GI: 1836111) and can be encoded by the nucleic acid sequenceset forth in NCBI Accession No. 577834.1 (GI: 999000). For example, ahuman CD19 can have the amino acid sequence set forth in NCBI AccessionNo. AAA69966.1 (GI: 901823) and can be encoded by the nucleic acidsequence set forth in NCBI Accession No. M84371.1 (GI: 901822). Forexample, a human CD20 can have the amino acid sequence set forth in NCBIAccession No. CBG76695.1 (GI: 285310157) and can be encoded by thenucleic acid sequence set forth in NCBI Accession No. AH003353.1 (GI:1199857). For example, a human CD80 can have the amino acid sequence setforth in NCBI Accession No. NP_005182.1 (GI: 4885123) and can be encodedby the nucleic acid sequence set forth in NCBI Accession No. NM_005191.3(GI: 113722122), and a human CD86 can have the amino acid sequence setforth in NCBI Accession No. AAB03814.1 (GI: 439839) and can be encodedby the nucleic acid sequence set forth in NCBI Accession No. CR541844.1(GI: 49456642). For example, a polypeptide that can be useful forregenerating cardiac function and/or tissue can be an antibody directedagainst TNF-α, mitochondrial complex-1, or resolvin-D1. In some cases, aparticle encapsulating a biologic described herein can encapsulate oneor more mRNAs encoding NAP-2 and/or TGF-α.

In some cases, a particle (e.g., an alginate gel) described herein canbe used to encapsulate one or more inhibitory RNAs useful to treat amammal experiencing a major adverse cardiac event (e.g., acutemyocardial infarction) and/or a mammal at risk of experiencing a majoradverse cardiac event (e.g., patients who underwent PCI for STEMI). Forexample, a particle described herein can encapsulate one or moreinhibitory RNAs inhibiting and/or reducing expression of one or more ofthe following polypeptides: eotaxin-3, cathepsin-S, DK-1, follistatin,ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI, TNFαRII,angiostatin, CCL25, ANGPTL4, MMP-3, and polypeptides described in WO2015/034897. For example, a human eotaxin-3 polypeptide can have anamino acid sequence set forth in, for example, NCBI Accession No: No.NP_006063.1 (GI No. 10344) and can be encoded by the nucleic acidsequence set forth in NCBI Accession No. NM_006072 (GI No. 10344). Insome cases, a human cathepsin-S can have the amino acid sequence setforth in NCBI Accession No. NP_004070.3 (GI No. 1520) and can be encodedby the nucleic acid sequence set forth in NCBI Accession No. NM_004079.4(GI No. 1520). In some cases, a human DK-1 can have the amino acidsequence set forth in NCBI Accession No. NP_036374.1 (GI No. 22943) andcan be encoded by the nucleic acid sequence set forth in NCBI AccessionNo. NM_012242 (GI No. 22943). In some cases, a human follistatin canhave then amino acid sequence set forth in NCBI Accession No.NP_037541.1 (GI No. 10468) and can be encoded by the nucleic acidsequence set forth in NCBI Accession No. NM_013409.2 (GI No. 10468). Insome cases, a human ST-2 can have the amino acid sequence set forth inNCBI Accession No. BAA02233 (GI No. 6761) and can be encoded by thenucleic acid sequence set forth in NCBI Accession No D12763.1 (GI No6761). In some cases, a human GRO-α polypeptide can have the amino acidsequence set forth in NCBI Accession No. NP_001502.1 (GI No. 2919) andcan be encoded by the nucleic acid sequence set forth in NCBI AccessionNo. NM_001511 (GI No. 2919). In some cases, a human IL-21 can have theamino acid sequence set forth in NCBI Accession No. NP_068575.1 (GI No.59067) and can be encoded by the nucleic acid sequence set forth in NCBIAccession No. NM_021803 (GI No. 59067). In some cases, a human NOVpolypeptide can have the amino acid sequence set forth in NCBI AccessionNo. NP_002505.1 (GI No. 4856) and can be encoded by the nucleic acidsequence set forth in NCBI Accession No. NM_002514 (GI No. 4856). Insome cases, a human transferrin polypeptide can have the amino acidsequence set forth in NCBI Accession No. NP_001054.1 (GI No. 7018) andcan be encoded by the nucleic acid sequence set forth in NCBI AccessionNo. NM_001063.3 (GI No. 7018). In some cases, a human TIMP-2 polypeptidecan have the amino acid sequence set forth in NCBI Accession No.NP_003246.1 (GI No. 7077) and can be encoded by the nucleic acidsequence set forth in NCBI Accession No. NM_003255.4 (GI No. 7077). Insome cases, a human TNFαRI polypeptide can have the amino acid sequenceset forth in NCBI Accession No. NP_001056.1 (GI No. 7132) and can beencoded by the nucleic acid sequence set forth in NCBI Accession No.NM_001065 (GI No. 7132). In some cases, a human TNFαRII polypeptide canhave the amino acid sequence set forth in NCBI Accession No. NP_001057.1(GI No. 7133) and can be encoded by the nucleic acid sequence set forthin NCBI Accession No. NM_001066 (GI No. 7133). In some cases, a humanangiostatin polypeptide can have the amino acid sequence set forth inNCBI Accession No. NP_000292 (GI No. 5340) and can be encoded by thenucleic acid sequence set forth in NCBI Accession No. NM_000301 (GI No.5340). In some cases, a human CCL25 polypeptide can have the amino acidsequence set forth in NCBI Accession No. NP_005615.2 (GI No. 6370) andcan be encoded by the nucleic acid sequence set forth in NCBI AccessionNo. NM_005624 (GI No. 6370). In some cases, a human ANGPTL4 polypeptidecan have the amino acid sequence set forth in NCBI Accession No.NP_001034756.1 or NP_647475.1 (GI No. 51129) and can be encoded by thenucleic acid sequence set forth in NCBI Accession No. NM_001039667.1 orNM_139314.1 (GI No. 51129). In some cases, a human MMP-3 polypeptide canhave the amino acid sequence set forth in NCBI Accession No. NP_002413.1(GI No. 4314) and can be encoded by the nucleic acid sequence set forthin NCBI Accession No. NM_002422 (GI No. 4314).

In some cases, a particle (e.g., an alginate gel) described herein canbe used to encapsulate one or more nucleotides that modulate (e.g.,mimic or inhibit) microRNAs involved in cardiac regenerative potency.For example, a particle described herein can be used to encapsulate oneor more agomiRs that mimic one or more miRNAs to augment cardiacregenerative potency. For example, a particle described herein can beused to encapsulate one or more antagomiRs that inhibit one or moremiRNAs to augment cardiac regenerative potency. Examples of miRNAsinvolved in cardiac regenerative potency include, without limitation,miR-127, miR-708, miR-22-3p, miR-411, miR-27a, miR-29a, miR-148a,miR-199a, miR-143, miR-21, miR-23a-5p, miR-23a, miR-146b-5p, miR-146b,miR-146b-3p, miR-2682-3p, miR-2682, miR-4443, miR-4443, miR-4521,miR-4521, miR-2682-5p, miR-2682, miR-137.miR-137, miR-549.miR-549,miR-335-3p, miR-335, miR-181c-5p, miR-181c, miR-224-5p, miR-224,miR-3928, miR-3928, miR-324-5p, miR-324, miR-548h-5p, miR-548h-1,miR-548h-5p, miR-548h-2, miR-548h-5p, miR-548h-3, miR-548h-5p,miR-548h-4, miR-548h-5p, miR-548h-5, miR-4725-3p, miR-4725, miR-92a-3p,miR-92a-1, miR-92a-3p, miR-92a-2, miR-134, miR-134, miR-432-5p, miR-432,miR-651, miR-651, miR-181a-5p, miR-181a-1, miR-181a-5p, miR-181a-2,miR-27a-5p, miR-27a, miR-3940-3p, miR-3940, miR-3129-3p, miR-3129,miR-146b-3p, miR-146b, miR-940, miR-940, miR-484, miR-484, miR-193b-3p,miR-193b, miR-651, miR-651, miR-15b-3p, miR-15b, miR-576-5p, miR-576,miR-377-5p, miR-377, miR-1306-5p, miR-1306, miR-138-5p, miR-138-1,miR-337-5p, miR-337, miR-135b-5p, miR-135b, miR-16-2-3p, miR-16-2,miR-376c.miR-376c, miR-136-5p, miR-136, let-7b-5p, let-7b, miR-3′7′7-3p,miR-377, miR-1273g-3p, miR-1273g, miR-34c-3p, miR-34c, miR-485-5p,miR-485, miR-370.miR-370, let-7f-1-3p, let-7f-1, miR-3679-5p, miR-3679,miR-20a-5p, miR-20a, miR-585.miR-585, miR-3934, miR-3934, miR-127-3p,miR-127, miR-424-3p, miR-424, miR-24-2-5p, miR-24-2, miR-130b-5p,miR-130b, miR-138-5p, miR-138-2, miR-769-3p, miR-769, miR-1306-3p,miR-1306, miR-625-3p, miR-625, miR-193a-3p, miR-193a, miR-664-5p,miR-664, miR-5096.miR-5096, let-7a-3p, let-7a-1, let-7a-3p, let-7a-3,miR-15b-5p, miR-15b, miR-18a-5p, miR-18a, let-7e-3p, let-7e,miR-1287.miR-1287, miR-181c-3p, miR-181c, miR-3653, miR-3653,miR-15b-5p, miR-15b, miR-1, miR-1-1, miR-106a-5p, miR-106a,miR-3909.miR-3909, miR-1294.miR-1294, miR-1278, miR-1278, miR-629-3p,miR-629, miR-340-3p, miR-340, miR-200c-3p, miR-200c, miR-22-3p, miR-22,miR-128, miR-128-2, miR-382-5p, miR-382, miR-671-5p, miR-671,miR-27b-5p, miR-27b, miR-335-5p, miR-335, miR-26a-2-3p, miR-26a-2,miR-376b.miR-376b, miR-378a-5p, miR-378a, miR-1255a, miR-1255a,miR-491-5p, miR-491, miR-590-3p, miR-590, miR-32-3p, miR-32, miR-766-3p,miR-766, miR-30c-2-3p, miR-30c-2, miR-128.miR-128-1, miR-365b-5p,miR-365b, miR-132-5p, miR-132, miR-151b.miR-151b, miR-654-5p, miR-654,miR-374b-5p, miR-374b, miR-376a-3p, miR-376a-1, miR-376a-3p, miR-376a-2,miR-149-5p, miR-149, miR-4792.miR-4792, miR-1.miR-1-2, miR-195-3p,miR-195, miR-23b-3p, miR-23b, miR-127-5p, miR-127, miR-574-5p, miR-574,miR-454-3p, miR-454, miR-146a-5p, miR-146a, miR-7-1-3p, miR-7-1,miR-326.miR-326, miR-301a-5p, miR-301a, miR-31′73-5p, miR-3173,miR-450a-5p, miR-450a-1, miR-7-5p, miR-7-1, miR-7-5p, miR-7-3,miR-450a-5p, miR-450a-2, miR-1291, miR-1291, miR-7-5p, miR-7-2, andmiR-17-5p, miR-17.

Nucleotides (e.g., RNA) encapsulated within a particle described hereincan be modified nucleotides. In some cases, nucleotides can be modifiedfor increased stability. For example, one or more uracil residues of anRNA described herein can be replace with a modified uracil residue.Examples of modified uracil residues include, without limitation,pseudouridine (T), dihydrouridine (D), and dideoxyuracil. An mRNA may bemodified to form a biofunctionalized microencapsulated modified mRNA(M³RNA), which are described in more detail below.

A particle (e.g., an alginate gel) described herein can be used toencapsulate other molecules in addition to or in place of a biologic. Insome cases, a particle (e.g., an alginate gel) described herein can beused to encapsulate one or more small molecules. For example, a particledescribed herein can be used to encapsulate one or more small moleculesuseful to treat a mammal experiencing a major adverse cardiac event(e.g., acute myocardial infarction) and/or a mammal at risk ofexperiencing a major adverse cardiac event (e.g., patients who underwentPCI for STEMI). For example, a particle described herein can encapsulateone or more small molecules useful for regenerating cardiac functionand/or tissue. Examples of small molecules useful for regeneratingcardiac function and/or tissue include, without limitation,lipopolysaccharides, tumor necrosis factor (e.g., TNF-α) antagonists,mitochondrial complex-1 antagonists, and resolvin-D1 agonists. In somecases, a particle (e.g., an alginate gel) described herein can be usedto encapsulate one or more microvesicles and/or exosomes. In some cases,a particle described herein can be used to encapsulate one or moremicrovesicles and/or exosomes useful to treat a mammal experiencing amajor adverse cardiac event (e.g., acute myocardial infarction) and/or amammal at risk of experiencing a major adverse cardiac event (e.g.,patients who underwent PCI for STEMI). For example, a particle describedherein can encapsulate one or more microvesicles and/or exosomes usefulfor regenerating cardiac function and/or tissue. Examples ofmicrovesicles and exosomes useful for regenerating cardiac functionand/or tissue include, without limitation, microvesicles and exosomesisolated from plasma, blood-derived products, and cultured stem cells.

A particle (e.g., an alginate gel) described herein also can include oneor more detectable labels. A detectable label can be incorporated intothe particle or encapsulated within the particle. Examples of detectablemolecules include, without limitation, bioluminescent label (e.g.,luciferase), fluorescent molecules (e.g., GFP and mCherry), andradionuclide molecules. In some cases, an mRNA expressing a detectablelabel is encapsulated within a particle such that temporal and/orspatial delivery of the encapsulated RNA can be monitored in a mammal.

A particle (e.g., an alginate gel) described herein also can include oneor more additional therapeutic molecules. A therapeutic molecule can beconjugated to a particle, embedded within a particle, encapsulatedwithin a particle, or any combination thereof. Examples of therapeuticagents include, without limitation, stem cells (e.g., mesenchymal stemcells, cardiac stem cells, and bone marrow), pharmaceuticals (e.g.,statins, analgesics, chemotherapeutics, beta blockers, antibiotics, andnutrients (e.g., carbohydrates, fats, vitamins, and minerals).

In some cases, a particle (e.g., an alginate gel) encapsulating one ormore biologics described herein can be used to encapsulate one or morenucleotides useful for treating other diseases and/or conditions.

Microencapsulated Modified mRNA (M³RNA)

M³RNA is a unique platform by which to induce rapid expression ofencoded genes into a broad array of tissues. In the context of the M³RNAplatform, M³RNA refers to modified microencapsulated mRNA; nakedmodified RNA (unencapsulated) is referred to as M²RNA. M³RNA is wellsuited for generating antibodies against new infectious threats.Intrinsic to this technological platform is the ability to rapidly scalewithin a short timeframe and simultaneously deliver multiple geneconstructs. Due to the use of mRNA as the driving biologics within thisplatform, there is no integrative or mutation risk with therapy.Furthermore, unlike AAV and other viral gene-delivery technologies, theM³RNA platform avoids risk of immune reaction to the delivery systemallowing its repetitive use with different constructs. M³RNA can bereadily evolved into an M³RNA-Ig delivery system allowing efficient,rapid and sustained expression of antibodies against putative pathogensfollowing delivery.

M³RNA has been tested to deliver several reporter and therapeutic geneconstructs in both in vitro and in vivo models. Further, M³RNA canaccommodate multi-gene therapeutic capability. M³RNA can simultaneouslydeliver, for example, several cardioregenerative genes in the setting ofacute myocardial infarction. Specific to the transformation of M³RNAtowards an M³RNA-Ig platform, is the demonstration of simultaneousdelivery of reporter genes that emulate the size of IgG heavy and lightchains in the form of GFP/mCherry mRNA (720 bp) and Firefly Luciferase(FLuc) mRNA (1653 bp). The system has been scaled to a three-genedelivery platform in vivo demonstrating similar penetrance for allreporter genes tested. The microencapsulated modified messenger RNAachieves rapid and robust protein expression within multiple cell linesand primary cells (human dermal fibroblasts, human cardiac fibroblasts,HEK293 cells, HL-1 cardiomyocytes, HUVAC cells, neonatal ratcardiomyocytes, and neonatal rat skeletal muscle cells). Furthermore,microencapsulating M³RNA using, for example, PEGylated chargednanoparticles induce rapid (within two hours) in vivo expressionfollowing direct injection into multiple organ systems (including bothskeletal and cardiac muscle) in murine and porcine models.

Kinetics of M²RNA (modified mCherry mRNA) transfected into HEK293 (humanembryonic kidney) cells resulted in mCherry protein expression in asearly as two hours. Daily quantification of expression yieldedfluorescence images for HEK293 cells at indicated time periods are shownin FIG. 16 showing a rapid, robust, and sustained protein expressionupon mRNA transfection for up to six days. Quantification offluorescence intensity within these cells (>10 fields of cells/timeperiod) documented increasing fluorescence intensity in the initial 24hours, sustained up to six days. Using flow cytometry as the goldstandard for quantitation of transfection efficiency, analysis ofmCherry protein expression levels at four hours and 24 hourspost-transfection was performed. Scatter plots, with the fluorescenceintensity on the x-axis and sideward scattering signal on the y-axis,revealed the consistent bimodal population upon transfection (FIG. 16)with the transition revealing the number of transfected cells as seen atfour hours and 24 hours documenting a transfection efficiency ofapproximately 95%.

The M³RNA platform is compatible with “hard-to-transfect” primary cellphenotypes such as, for example, neonatal rat cardiomyocytes. Neonatalprimary cardiomyocytes were isolated and plated yielding synchronousbeating pattern of cardiomyocytes in the dishes. Cardiomyocytes weretransfected with mCherry M³RNA and fluorescence images were acquiredstarting at four hours post-transfection for six days days.Representative images for multiple time periods are shown in FIG. 17A,indicating rapid, robust, and sustained protein expression withinprimary cardiomyocytes. Quantification of the fluorescence intensitywithin these primary cardiomyocytes revealed that maximum expressionoccurred at 24 hours and was sustained, but declining, for up to sixdays (FIG. 17B). Transfection efficiency within these primarycardiomyocytes was furthermore assessed using flow cytometry at fourhours and 24 hours by transfecting the cells with mCherry mRNA withscatter plot analysis demonstrating a transfection efficiency of 43% at24 hours (FIG. 17C). Transfection of primary cardiomyocytes the M³RNAplatform did not alter the structural or functional characteristics ofcardiomyocytes.

The M³RNA platform also is compatible with intramuscular delivery,providing high transfection efficiency comparable with results obtainedwith primary cardiomyocyte cultures (FIG. 19). The data presented inFIG. 19 employ a primary human skeletal muscle culture system thatinvolves large-scale isolation of satellite cells from surgicallydisposed human skeletal muscle tissue. Within a defined xeno-freecultivating culture condition, skeletal muscle donor tissue is used toderive skeletal muscle satellite progenitor populations. Cells undergomycoplasma/sterility profiling over a three-week culture period and arecryopreserved. Prior to derivation of master cell banks, each lot ofcryopreserved satellite cells undergoes quality assurance evaluationtesting myogenic potency via immunohistochemistry (FIG. 18), geneexpression profiling and automated microscopic visualization of myotubeformation (FIG. 18). With confirmation of potency and sterility, eachlot is expanded in three passages (P3) to generate a master cell bank of200 million cells frozen as 1 million cell aliquots. Master cell banklots are subjected to repeat quality assurance and sterility assessmentprior to experimental use. P3 cells can be induced to generate primaryhuman skeletal muscle cells within 2-3 days through culture within adefined medium (FIG. 18).

Microencapsulation of modified messenger RNA within a PEGylatedcation-based micro particle system provides an exemplary platform bywhich to deliver M³RNA in vivo. Reporter genes including mCherry (720bp), GFP (720 bp) mRNA, and Firefly Luciferase (FLuc) mRNA (1653 bp)were used in the in vivo setting. FLuc provided an added dimension tothe analysis as the kinetics of protein expression in live animals couldbe prospectively documented. Using Luciferin to excite transfectionareas provided an exact approach to decipher the localization of theM³RNA signal following delivery. Following optimization using asubcutaneous route, the M³RNA platform was tested in a broad array oftissues including intrahepatic, intrarenal, intraocular, intramuscularand intramyocardial delivery (FIG. 20). In every instance, FLucexpression could be demonstrated within the injected area within twohours following delivery and sustained for more than 72 hours.

Within the heart, rapid FLuc expression following direct myocardialinjections of FLuc M³RNA into the anterior left ventricle was quantifiedat different time points, revealing rapid induction of gene expressionsustained over a three-day observation period (FIG. 21). To demonstrateas a proof-of-concept that multiple gene induction is feasible, threedifferent model constructs (mCherry, GFP, and FLuc) were simultaneouslymicroencapsulated and delivered using a single myocardial injection inmice versus scrambled mock M³RNA controls. FLuc expression was confirmedat 24 hours within the heart using the bioluminescence assay in IVISspectrum in vivo imaging system (FIG. 20, heart). Mice were thensacrificed and the heart was excised to assess expression byimmunohistochemistry in histological sections confirming overlappingGFP, mCherry, and FLuc protein expression within the combined gene-M³RNAinjected mice (lower panels) when compared to mock M³RNA mice (upperpanels) (FIG. 22).

The M³RNA technology can be evolved to achieve a delivery platform forrapid creation of highly potent therapeutics. For example,biofunctionalized M³RNA can form the foundation of an M³RNA-Ig DeliverySystem (MIDS) capable of rapidly integrating new genetic sequences totarget novel pathogens (e.g., viral pathogen such as, for example, Zikavirus and H7N9).

Compatibility of the M³RNA platform for the gene length of IgG heavy andlight chain has been confirmed with the concomitant use of FLuc andfluorescent reporter gene, showing simultaneous induction of up to threegenes within cardiac muscle tissue in vivo. Rapid and sustainableexpression of genes encoded in the M³RNA-Ig vector can be enhanced usingspecialized design of the 5′ and 3′ UTR of the RNA molecule. Design ofthe M³RNA-Ig vector can involve consideration of one or more of thefollowing: (1) whether to join the heavy and light chain genes on asingle transcript with a P2A ribosomal skipping sequence versussynthesis as separate transcripts; (2) the length/introduction of the3′poly(A) tail; (3) the type of 5′m7G cap; (4) selection of modifiednucleotides; (5) IRES and pseudoknot modification of the 5′ and 3′ UTR,respectively, to diminish rate of degradation; and/or (6)nanoparticle-mediated microencapsulation to identify an appropriatestoichiometry for in vivo skeletal muscle delivery.

In some embodiments, the heavy and light chains can be synthesized ontwo separate transcripts. This approach allows rapid parallel work toincorporate the VL/VH antigen binding domains within a multicloning site(FIG. 23). Specifically, using two transcripts instead of one allows theseparate DNA templates to be cloned at the same time instead ofsequentially, thereby speeding development of new embodiments. Ofcourse, where desired, one can design the vector so that the heavy chainand light chain are synthesized from a single transcript.

mRNAs synthesized with poly(A) tail of 300 or more nucleotides havesignificant longer half-life and superior translational properties. Insome embodiments, therefore, the vector can include a suitable promoter(e.g., T7), the heavy chain and light chain coding regions, and 300nucleotide poly(T) tract will serve as the backbone template for mRNAtranscription. Vectors can be designed as two separate versions toaccommodate large variances in the C-regions of new antibodiesidentified. The initial design can assume an unchanging C-region inprospective antibodies identified. Accordingly, the multi-cloning sitein the heavy chain and light chain vectors can allow rapid parallelintroduction of the V sequence into this region and initiation of theM³RNA-Ig manufacturing process. Conversely, in certain cases, a variedIg backbone may suggest adjustment also to the C regions of the parallelvector systems. As such, to ensure the capability for rapid response,additional vectors engineered with multi-cloning sites allowing forinsertion of the entirety of the heavy and light chains can be designed.

The mRNA CAP mediates efficiency of the expression of the gene product.In some embodiments, M³RNA-Ig may be generated using CAP-1 as anapproach to avoid any innate immune responses that may be mediated usingCAP-0. A design including CAP-1 can include post-transcriptionalmodifications using a Vaccinia Capping System followed by a CAP 2′O-Methyltransferase. However, a CAP-dependent approach can involveinteraction of the poly(A) binding protein (PABP), which can addsignificant length to the construct and thereby limit creativity at the3′ site to further limit RNA degradation (FIG. 24, top). Thus, in someembodiments, the M³RNA-Ig system can include an integral ribosomal entrysite (IRES) within the 5′UTR, eliminating the need for PABP interaction(FIG. 24, bottom).

An M³RNA system can limit undesirable side effects of mRNA transfectionand/or slow degradation of the M³RNA by introducing modified nucleotidessuch as, for example, 5′-methylcytidine in place of cytosine orpseudouridine (T), dihydrouridine (D), or dideoxyuracil in place ofuracil. Modified NTPs are readily abundant as GMP starting material andcan be rapidly introduced using standard RNA synthesis techniques,providing significant molecular and translational advantage followingdelivery.

Alternatively, another strategy for extending the life of an mRNA in thecytosol involves interfering with the nonsense-mediated decay pathway.In the canonical pathway, as the ribosome complex hits the gag stopregion, the 30s/50s subunits disengage from the mRNA, thus rendering the3′ UTR of the mRNA highly susceptible to a UPF-1 mediated decay (FIG.25, Canonical). Approaches to slow this process have been developed bydifferent viruses including, for example, 3′ pseudoknot formation afterthe gag stop site or introduction of an RNA stability element in the3′UTR. The 3′ pseudoknot mediates a frameshift read-through by theribosomal complex, thereby knocking UPF-1 complexes off of the 3′UTR,resulting in mRNA stability (FIG. 25, Pseudoknot). The RNA stabilityelement on the other hand, serves as a decoy, directly blocking UPF-1interaction with the 3′UTR, and thereby stabilizing mRNA against decay(FIG. 25, RNA Stability Element). Poly(A) stem loop structures also canincrease mRNA stability by inhibiting exosome-mediated mRNA degradation(FIG. 25, Stem Loop).

Nanoparticles for Delivery of Encapsulated or Unencapsulated mRNAs

Design of efficient gene delivery vectors possessing the hightransfection efficiencies and low cytotoxicity is one challenge fordelivering modified mRNAs—e.g., M²RNA, M³RNA, or M³RNA-Ig) to cells,tissue, or organs. Nanoparticles represents an exemplary model viralvector-free approach to deliver modified mRNAs. Nanoparticles possessremarkable flexibility for gene delivery including tissue targeting,protect mRNA against nuclease degradation, improve M²RNA stabilitythrough ionic interactions between the negatively charged mRNA andpositively charged nanoparticle surface, and increase transformationefficiency for safety. Nanoparticles are generally accepted fortherapeutic applications. First, nanoparticles exist in the same sizedomains as proteins. Second, nanoparticles have large surface areas thatcan be easily modified using, for example, PEGylation (to increase bloodcirculation half-life), or a poloxamer, a poloxamine, or chitosan forefficient binding and delivery. Third, modified nanoparticles can havecontrollable absorption and release properties, particle size, and/orsurface characteristics.

A wide variety of organic (lipid-based), inorganic, or hybrid materialsare used to produce nanoparticles and are discussed in detail above. Insome embodiments, cationic polymer nanoparticles are used tomicroencapsulate modified messenger RNA. Cationic polymers havepositively charged groups in their backbone to interact with negativelycharged mRNA-Ig molecules to form neutralized, nanometer-sizedcomplexes.

Multiple metallic nanoparticles have been suggested to cause minimumcytotoxicity. In various embodiments, nanoparticles made of iron,silver, gold, or copper can be used, alone or in combination with othernanoparticles and/or other delivery technologies, for delivering mRNA-Igmolecules.

Nanoparticles may be surface modified to increase the efficiency ofmodified RNA molecules, whether M²RNA (or M²RNA-Ig) or M³RNA (orM³RNA-Ig). Nanoparticles may be modified to introduce, for example,either a biopolymer or PEGylation to increase blood circulationhalf-life. In some embodiments for the delivery of M³RNA-Ig to skeletalmuscle, the surface of the nanoparticle may be modified to include abiopolymer. Suitable biopolymers include, for example, collagen,elastin, fibronectin, chitosan, dextran etc. In particular embodiments,the surface of the nanoparticle may be modified with chitosan. Chitosanexhibits a cationic polyelectrolyte nature and therefore provides astrong electrostatic interaction with negatively charged DNA or RNAmolecules. Moreover, chitosan carries primary amine groups that makes ita biodegradable, biocompatible, and non-toxic biopolymer that providesprotection against DNase or RNase degradation.

In alternative embodiments, the surface of the nanoparticles may bemodified by PEGylation. The technique of covalently attaching thepolyethylene glycol (PEG) to a given molecule, nanoparticle in thiscase, is a well-established method in targeted drug delivery systems.PEGylation involves the polymerization of multiple monomethoxy PEG(mPEG) that are represented as CH₃O—(CH₂—CH₂O)_(n)—CH₂—CH₂—OH.Introducing PEG molecules significantly increases the half-life of ananoparticle due to its increased hydrophobicity, reduces glomerularfiltration rate, and/or lowers immunogenicity due to masking ofantigenic sites by forming protective hydrophilic shield. Suitablemodifications include modifying the surface of the nanoparticles topossess 3000-4000 PEG molecules, which provides a suitable environmentfor the physical binding of DNA or RNA molecules.

Taken together, the combination of nanoparticles with PEG, chitosan, andM²RNA generate the M³RNA platform (FIG. 26). In the exemplary embodimentillustrated in FIG. 26, the modified mRNA (M²RNA) is encapsulated by thePEG-and-chitosan-coated microparticle since the RNA is indirectly boundto the microparticle through interaction with the chitosan surfacemodification.

Beyond molecular design, another challenge facing delivery of biologicsin muscle tissue is the physical barrier to efficient transfer ofbiologic payload due, at least in part, to the dense and contractilenature of muscle tissue. Four-dimensional modeling of delivery in tissueusing Darcy's Law, a significant limitation has been identified at alevel inherent to the design of the needle. By having a straight needlewith an end hole, biologics delivery creates a dense pocket of materialwithin skeletal muscle tissue that mimics an abscess. The dense pocketdramatically reduces local tissue uptake of the biologics andpreferentially eliminates the biologic via lymphatic and capillaryaction to alleviate the pressure. Conversely, an adjusted needle design,shown in FIG. 14, devoid of an end hole and utilizing a non-sheeringconduit pattern with the delivery needle allows the ability to induce anedema effect instead of an abscess, yielding a much higher degree oftissue accommodation and protracted local exposure of product to tissue.In this way, although the dose of the biologic is not increased thepenetrance of the therapy increases by an average of 4-5 fold.

Methods of Using

This document also provides methods of using a particle (e.g., analginate gel) encapsulating one or more molecules (e.g., biologics)described herein. In some cases, a mammal at risk of experiencing amajor adverse cardiac event (e.g., a mammal identified as being likelyto experience a major adverse cardiac event as described herein) can betreated by administering a particle encapsulating one or more biologicsdescribed herein. For example, a mammal at risk of experiencing a majoradverse cardiac event can be treated by administering a particleencapsulating mRNA encoding NAP-2, TGF-α, ErBb3, VEGF, IGF-1, FGF-2,PDGF, IL-2, CD19, CD20, and/or CD80/86 to increase the level of NAP-2,TGF-α, ErBb3, VEGF, IGF-1, FGF-2, PDGF, IL-2, CD19, CD20, and/or CD80/86polypeptide expression. An increase in the level of one or more of thesepolypeptides can be used to reduce scar size and tissue remodeling andto improve cardiac function. For example, a mammal at risk ofexperiencing a major adverse cardiac event can be treated byadministering a particle encapsulating an inhibitory RNA targetingeotaxin-3, cathepsin-S, DK-1, follistatin, ST-2, GRO-α, IL-21, NOV,transferrin, TIMP-2, TNFαRI, TNFαRII, angiostatin, CCL25, ANGPTL4,and/or MMP-3 to decrease the level of eotaxin-3, cathepsin-S, DK-1,follistatin, ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI,TNFαRII, angiostatin, CCL25, ANGPTL4, and/or MMP-3 polypeptideexpression. A decrease in the level of one or more of these polypeptidescan be used to reduce scar size and tissue remodeling and to improvecardiac function.

Any type of mammal experiencing a major adverse cardiac event (e.g.,acute myocardial infarction) and/or a mammal at risk of experiencing amajor adverse cardiac event (e.g., a mammal who underwent PCI for STEMI)can be treated with using particle-mediated delivery of one or morebiologics described herein. For example, humans and other primates suchas monkeys experiencing a major adverse cardiac event and/or at risk ofexperiencing a major adverse cardiac event can be treated with biologicsas described herein. In some cases, dogs, cats, horses, cows, pigs,sheep, rabbits, mice, and rats can be treated with biologics asdescribed herein.

Any appropriate method can be used to identify a mammal experiencing amajor adverse cardiac event (e.g., acute myocardial infarction) and/or amammal at risk of experiencing a major adverse cardiac event (e.g.,patients who underwent PCI for STEMI). For example, the methods ofidentifying a mammal at risk of experiencing a major adverse cardiacevent described elsewhere (e.g., WO 2015/034897) can be used.

Once identified as experiencing a major adverse cardiac event (e.g.,acute myocardial infarction) and/or being at risk of experiencing amajor adverse cardiac event, the patient can be administered orinstructed to self-administer one or more particles encapsulating amolecule (e.g., a biologic) as described herein.

When treating mammal experiencing a major adverse cardiac event or atrisk of experiencing a major adverse cardiac event as described herein,the major adverse cardiac event can be any major adverse cardiac event.Examples of major adverse cardiac events include, without limitation,myocardial infarction (e.g., acute myocardial infarction), heartfailure, recurrent myocardial infarction, repeat hospitalization forcardiac-related events, and ischemic heart disease. In some embodiments,the major adverse cardiac event treated as described herein can bemyocardial infarction, such as acute myocardial infarction.

In some cases, particle-mediated delivery of one or more molecules(e.g., biologics) to a mammal as described herein can be used to improvecardiac function. Examples of improved cardiac function include, withoutlimitation, increased survivorship, reduced hospitalization,symptom-free tolerance of physical activity, improved global physicalfitness, improved cardiac ejection fraction, improved cardiac output,improved stroke volume, improved cardiac mass index, and reduced scarsize.

In some cases, particle-mediated delivery of one or more biologics(e.g., mRNA) to a mammal as described herein can be used to increaseexpression of one or more (e.g., one, two, three, or more) polypeptidesuseful for regenerating cardiac function and/or tissue. Examples ofpolypeptides that can be useful for regenerating cardiac function and/ortissue include, without limitation, NAP-2, TGF-α, ErBb3, VEGF, IGF-1,FGF-2, PDGF, IL-2, CD19, CD20, CD80/86, and polypeptides described in WO2015/034897. An increase in the level of one or more of thesepolypeptides can be used to reduce scar size and tissue remodelingand/or improve cardiac function. Methods for increasing expression of apolypeptide useful for regenerating cardiac function and/or tissue incells (e.g., cardiomyocytes) can include contacting the cells with oneor more particles encapsulating, for example, an mRNA encoding thepolypeptide. One or more particles encapsulating an mRNA encoding thepolypeptide can be contacted with the cells by any appropriate method.The term “increased expression” as used herein with respect to the levelof a polypeptide is any level that is greater than (e.g., at least about10, 15, 20, or 25 percent greater than) a reference level for thatpolypeptide. The term “reference level” as used herein with respect to apolypeptide is the level of expression of that polypeptide typicallyobserved by healthy humans or humans with a low risk of experiencing amajor adverse cardiac event. For example, levels of NAP-2, TGF-α, andErBb3 expression with respect to healthy humans or humans with a lowrisk of experiencing a major adverse cardiac event can be as describedelsewhere (e.g., WO 2015/034897). In some cases, particle-mediateddelivery of one or more biologics (e.g., mRNA) to a mammal as describedherein can be used to increase expression of NAP-2 and/or TGF-α.

In some cases, particle-mediated delivery of one or more biologics(e.g., inhibitory RNA) to a mammal described herein can be used todecrease expression of one or more (e.g., one, two, three, four, five,six, seven, eight, nine, ten, or more) of the following polypeptides:eotaxin-3, cathepsin-S, DK-1, follistatin, ST-2, GRO-α, IL-21, NOV,transferrin, TIMP-2, TNFαRI, TNFαRII, angiostatin, CCL25, ANGPTL4,MMP-3, and polypeptides described in WO 2015/034897. A decrease in theexpression level of one or more of these polypeptides can be used toreduce scar size and tissue remodeling and/or improve cardiac function.Methods for decreasing expression of one or more of eotaxin-3,cathepsin-S, DK-1, follistatin, ST-2, GRO-α, IL-21, NOV, transferrin,TIMP-2, TNFαRI, TNFαRII, angiostatin, CCL25, ANGPTL4, and MMP-3 in cellscan include contacting the cells (e.g., cardiomyocytes) with one or moreparticles encapsulating, for example, an inhibitory RNA. One or moreparticles encapsulating an inhibitory RNA can be contacted with thecells by any appropriate method. For example, in humans, a particleencapsulating an inhibitory RNA described herein can be used to decreaseexpression of a human eotaxin-3, a human cathepsin-S, a human DK-1, ahuman follistatin, a human ST-2, a human GRO-α, a human IL-21, a humanNOV, a human transferrin, a human TIMP-2, a human TNFαRI, a humanTNFαRII, a human angiostatin, a human CCL25, a human ANGPTL4, a humanMMP-3, or any combination thereof. The term “decreased expression” asused herein with respect to the level of a polypeptide (e.g., eotaxin-3,cathepsin-S, DK-1, follistatin, ST-2, GRO-α, IL-21, NOV, transferrin,TIMP-2, TNFαRI, TNFαRII, angiostatin, CCL25, ANGPTL4, or MMP-3) is anylevel that is lower than (e.g., at least about 10 percent, at leastabout 15 percent, at least about 20 percent, or at least about 25percent lower than) a reference level for that polypeptide. The term“reference level” as used herein with respect to a eotaxin-3,cathepsin-S, DK-1, follistatin, ST-2, GRO-α, IL-21, NOV, transferrin,TIMP-2, TNFαRI, TNFαRII, angiostatin, CCL25, ANGPTL4, or MMP-3polypeptide is the level of expression of that polypeptide typicallyobserved by healthy humans or humans with a low risk of experiencing amajor adverse cardiac event. Levels of eotaxin-3, cathepsin-S, DK-1,follistatin, ST-2, GRO-a, IL-21, NOV, transferrin, TIMP-2, TNFαRI,TNFαRII, angiostatin, CCL25, ANGPTL4, and MMP-3 expression with respectto healthy humans or humans with a low risk of experiencing a majoradverse cardiac event can be as described elsewhere (e.g., WO2015/034897).

A particle (e.g., an alginate gel) encapsulating one or more molecules(e.g., biologics) described herein can be administered to a mammalexperiencing a major adverse cardiac event or likely to experience amajor adverse cardiac event as a combination therapy with one or moreadditional agents/therapies used to treat a major adverse cardiac event.For example, a combination therapy used to treat a mammal identified asbeing likely to experience a major adverse cardiac event as describedherein can include administering an alginate gel encapsulating one ormore biologics described herein and treating with aggressivepharmacotherapy (e.g., beta-adrenoceptor blockade, angiotensinconverting enzyme inhibitors, aldosterone antagonism treatments, and/orantiplatelet agents), hemodynamic support (e.g., intra-aortic balloonpump and/or mechanical augmentation of cardiac output), surgicalintervention (e.g., coronary bypass grafting or left ventricular assistdevice placement), and/or device-based intervention (e.g.,resychronization therapy or implantable cardiac defibrillators).

In embodiments where a particle (e.g., an alginate gel) encapsulatingone or more molecules (e.g., biologics) described herein is used incombination with additional agents/therapies used to treat a majoradverse cardiac event, the one or more additional agents can beadministered at the same time or independently. For example, an alginategel encapsulating one or more biologics described herein can beadministered first, and the one or more additional agents administeredsecond, or vice versa. In embodiments where a particle (e.g., analginate gel) encapsulating one or more biologics described herein isused in combination with one or more additional therapies used to treata major adverse cardiac event, the one or more additional therapies canbe performed at the same time or independently of the administration ofone or more particles encapsulating one or more biologics describedherein. For example, the one or more alginate gels encapsulating one ormore biologics described herein can be administered before, during, orafter the one or more additional therapies are performed.

In some cases, a particle (e.g., an alginate gel) encapsulating one ormore molecules (e.g., biologics) described herein can be used to treatnumerous other diseases and/or conditions including, without limitation,a hematologic disorder or hematologic malignancy (e.g., lymphoma,lymphocytic leukemia, myeloma, myelogenous leukemia (e.g., acutemyelogenous leukemia and chronic myelogenous leukemia), myelodysplasticsyndromes, and myeloproliferative diseases), a musculoskeletal disorder(e.g., carpal tunnel syndrome, epidondylitis, tendinitis, back pain,tension neck syndrome, and hand-arm vibration syndrome), a pulmonarycondition (e.g., asthma, chronic obstructive pulmonary disease, chronicbronchitis, emphysema, acute bronchitis, cystic fibrosis, pneumonia,tuberculosis, emphysema, pulmonary edema, acute respiratory distresssyndrome, pneumoconiosis, and interstitial lung disease), agastrointestinal, colorectal, anal-sphincter and/or pelvic organ disease(e.g., enterocolitis, infectious diarrhea, mesenteric ischaemia,inflammatory bowel disease, and pelvic inflammatory disease), aneurologic, spinal cord and/or intracranial disease (e.g., neural tubedefects, cephalic disorders, raised or decreased intracranial pressure,meningitis, neuropathies, motor neuron diseases, demyelinatingneuropathies, and nerve injuries), a dermatologic disorder (e.g., eczema(e.g., atopic dermatitis), warts, acne, and roseola), chronicinflammatory conditions (e.g., asthma, chronic peptic ulcer,tuberculosis, rheumatoid arthritis, chronic periodontitis, ulcerativecolitis and crohn's disease, chronic sinusitis, chronic activehepatitis), and/or genetic defects.

In some cases, a particle (e.g., an alginate gel) encapsulating one ormore molecules (e.g., biologics) described herein can be formulated intoa pharmaceutically acceptable composition for administration to a mammalexperiencing a major cardiac event or at risk of experiencing a majorcardiac event. For example, a therapeutically effective amount of analginate gel encapsulating a biologic described herein can be formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. A pharmaceutical composition can beformulated for administration in solid or liquid form including, withoutlimitation, sterile solutions, suspensions, sustained-releaseformulations, tablets, capsules, pills, powders, and granules.

Pharmaceutically acceptable carriers, fillers, and vehicles that may beused in a pharmaceutical composition described herein include, withoutlimitation, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A pharmaceutical composition containing a particle (e.g., an alginategel) encapsulating one or more molecules (e.g., biologics) describedherein can be designed for oral, parenteral (including subcutaneous,intraarterial, intramuscular, intravenous, intracoronary, intradermal,or topical), or inhaled administration. When being administered orally,a pharmaceutical composition containing a particle (e.g., an alginategel) encapsulating one or more biologics described herein can be in theform of a pill, tablet, or capsule. Compositions suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions that can contain anti-oxidants, buffers, bacteriostats, andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents. Compositionsfor inhalation can be delivered using, for example, an inhaler, anebulizer, and/or a dry powder inhaler. The formulations can bepresented in unit-dose or multi-dose containers, for example, sealedampules and vials, and may be stored in a freeze dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets.

A pharmaceutically acceptable composition including a particle (e.g., analginate gel) encapsulating one or more molecules (e.g., biologics)described herein can be administered locally or systemically. In somecases, a composition containing a particle encapsulating one or morebiologics described herein can be administered systemically by venous ororal administration to, or inhalation by a mammal (e.g., a human). Insome cases, a composition containing a particle encapsulating one ormore biologics described herein can be administered locally bypercutaneous, subcutaneous, intramuscular, or open surgicaladministration (e.g., injection) to a target tissue (e.g., cardiacinfarct bed) of a mammal (e.g., a human), or by arterial administrationto the vascular supply of a target tissue (e.g., cardiac infarct bed) ofa mammal (e.g., a human). For example, arterial administration of analginate gel encapsulating one or more biologics described herein to thevascular supply of the heart can be used to deliver the biologics to thecardiac infarct bed of a human.

Effective doses can vary depending on the severity of the major cardiacevent, the route of administration, the age and general health conditionof the subject, excipient usage, the possibility of co-usage with othertherapeutic treatments such as use of other agents, and the judgment ofthe treating physician.

The frequency of administration can be any frequency that improvescardiac function without producing significant toxicity to the mammal.For example, the frequency of administration can be from about once aweek to about three times a day, from about twice a month to about sixtimes a day, or from about twice a week to about once a day. Thefrequency of administration can remain constant or can be variableduring the duration of treatment. A course of treatment with acomposition containing a particle (e.g., an alginate gel) encapsulatingone or more biologics described herein can include rest periods. Forexample, a composition containing an alginate gel encapsulating one ormore biologics described herein can be administered daily over atwo-week period followed by a two week rest period, and such a regimencan be repeated multiple times. As with the effective amount, variousfactors can influence the actual frequency of administration used for aparticular application. For example, the effective amount, duration oftreatment, use of multiple treatment agents, route of administration,and severity of the major cardiac event may require an increase ordecrease in administration frequency.

An effective duration for administering a composition containing aparticle (e.g., an alginate gel) encapsulating one or more molecules(e.g., biologics) described herein can be any duration that improvescardiac function without producing significant toxicity to the mammal.For example, the effective duration can vary from several days toseveral weeks, months, or years. In some cases, the effective durationfor the treatment of a major cardiac event can range in duration fromabout one month to about 10 years. Multiple factors can influence theactual effective duration used for a particular treatment. For example,an effective duration can vary with the frequency of administration,effective amount, use of multiple treatment agents, route ofadministration, and severity of the major cardiac event being treated.

In certain instances, a course of treatment and the cardiac function ofthe mammal being treated for a major cardiac event can be monitored. Anyappropriate method can be used to monitor cardiac function. For example,cardiac function can be assessed using blood tests, electrocardiography(ECG/EKG), cardiac stress testing, coronary catheterization,echocardiogram, and/or intravascular ultrasound at different timepoints.

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Examples Example 1: mRNA Expression in Fibroblasts

mCherry mRNA was transfected into human dermal fibroblasts and humancardiac fibroblasts. Light phase and fluorescent microscopy was used toevaluate mRNA expression at 4 hours, 2 days, and 5 days posttransfection (FIG. 3).

These results demonstrated that mRNA can be sustainably expressed indermal and cardiac fibroblasts.

Example 2: mRNA Co-Expression in Epithelial Cells

mCherry mRNA and EGFP mRNA were transfected into HEK293 cells.Multi-channel fluorescent microscopy was used to evaluate mRNAexpression at 24 hours post transfection (FIG. 4).

These results demonstrated that multiple mRNAs can be co-expressed.

Example 3: mRNA Expression in Cardiomyocytes

mCherry mRNA was transfected into HEK293 cells. Light phase andfluorescent microscopy was used to evaluate mRNA expression at 24 hours,3 days, and 5 days post transfection (FIG. 5).

These results demonstrated that mRNA can be sustainably expressed incardiomyocytes.

Example 4: In Vivo Expression of Subcutaneous Liposome-Delivered mRNA

Mice were administered a solution of luciferase mRNA via hydrodynamictail vein injection (FIGS. 6(A) and (B)) or administered liposomescontaining luciferase mRNA via subcutaneous injection (FIGS. 6(C) and(D)). Luciferase expression was imaged using a Xenogen (IVIS) imagingsystem. For mice administered a solution of luciferase mRNA viahydrodynamic tail vein injection, the amount of luciferase expressed wasevaluated at the beginning of the experiment and at two hours, fourhours, six hours, and 24 hours after administration (FIG. 6E). For miceadministered liposomes containing luciferase mRNA via subcutaneousinjection, the amount of luciferase expressed was evaluated at twohours, four hours, six hours, 24 hours, 48 hours, and 72 hours afteradministration (FIG. 6F).

Mice were administered liposomes containing mCherry mRNA viasubcutaneous injection. mCherry expression was evaluated usingfluorescent microscopy (FIG. 7).

These results demonstrated that liposome delivered mRNA can besustainably expressed in vivo following subcutaneous administration.

Example 5: In Vivo Expression of Intracardiac Liposome-Delivered mRNA

Mice were administered liposomes containing luciferase mRNA via echoguided intracardiac injection (FIG. 8A). Luciferase expression wasimaged using a Xenogen (IVIS) imaging system. The amount of luciferaseexpressed was evaluated at 2, 4, 6, 24, 48, and 72 hours afteradministration (FIG. 8B).

These results demonstrated that liposome delivered mRNA can besustainably expressed in vivo following intracardiac administration.

Example 6: In Vivo Expression of Open-Chest IntracardiacLiposome-Delivered mRNA

Mice were administered liposomes containing luciferase mRNA via openchest intracardiac injection. Luciferase expression was imaged using aXenogen (IVIS) imaging system (FIG. 9).

These results demonstrated that liposome delivered mRNA can besustainably expressed in vivo following intracardiac administration.

Example 7: In Vivo Expression of Alginate-Delivered mRNA

Pigs were administered alginate gel containing mCherry mRNA usingprevious reported approaches. The amount of mCherry expression wasevaluated after the administration (FIG. 10).

Previous methods of using alginate delivery of mRNA resulted insequestration of biologics within the polymerized product with nointramyocardial expression.

Example 8: In Vivo Expression of Alginate-Delivered mRNA

Pigs were administered a reduced volume of alginate gel containingmCherry mRNA. mCherry expression was evaluated using fluorescentmicroscopy.

Reduced alginate gel volume resulted in diffuse delivery of biologicsand loss of the majority of signal (3.6 radiance efficiency; FIG. 11).

Example 9: In Vivo Expression of Alginate-Delivered mRNA

Pigs were administered alginate gels having varied alginate/calciumconcentrations containing mCherry mRNA. mCherry expression was evaluatedusing fluorescent microscopy.

Targeted delivery into the infarct bed with an alginate/calcium gelremaining liquid in blood and escaping into the infarcted capillary bedwhere it polymerized and delivered the biologics with high efficiency(localized radiance level >10 versus 3-4 in prior efforts; FIG. 12).

These results demonstrated that alginate gel delivered mRNA can betargeted to infarcted capillary beds.

Example 10: In Vivo Expression of Open-Chest IntracardiacAlginate-Delivered mRNA

Mice were administered alginate gels having varied alginate/calciumconcentrations containing mCherry mRNA. mCherry expression was evaluatedusing fluorescent microscopy.

Alginate gel delivery resulted in sustained release of mCherry mRNA(FIG. 13).

These results demonstrated that alginate gel delivered mRNA can besustainably expressed in vivo following delivery.

Sequence Listing Free Text SEQ ID NO: 1CCCATTGTAT GGGATCTGAT CTGGGGCCTC GGTGCACATGCTTTACATGT GTTTAGTCGA GGTTAAAAAA SEQ ID NO: 2ACGTCTAGGC CCCCCGAACC ACGGGGACGT GGTTTTCCTTT GAAAAA SEQ ID NO: 3CCAGAAGGTA CCCCATTGTA TGGGATCTGA TCTGGGGCCTCGGTACACAT GCTTTACATG TGTTTAGTCG AGGTTAAAAAAACGTCTAGG CCCCCCGAAC CACGGGGACG TGGTTTTCCT TTGAAAAACA CGATGATASEQ ID NO: 4 ATATGGCCAC AACCATGGTG AGCAAGGGCG AGGAGCTGTTCACCGGGGTG GTGCCCATCC TGGTCGAGCT GGACGGCGACGTAAACGGCC ACAAGTTCAG CGTGTCCGGC GAGGGCGAGGGCGATGCCAC CTACGGCAAG CTGACCCTGA AGTTCATCTG CACCACCGGC AAG SEQ ID NO: 5GCTGCTGCCC GACAACCACT ACCTGXAGCX ACCCAGTCCGCCCTGAGCAA AGACCCCAAC GAGAAGCGCG ATCACATGGTCCTGCTGGAG TTCGTGACCG CCGCCGGGAT CACTCTCGGCATGGACGAGC TGTACAAGTA AGCCCTGTGG AATGTGTGTCAGTTAGXXXG GTGTGGAAAG TCCCCAXXGG CTCCCCXXXAGCAXXXXXXX XXXXXGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGSEQ ID NO: 6 CAACGTCTAT ATCATGGCCG ACAAGCAGAA GAACGGCATCXXXAAGGTGA ACTTCAAGAT CCGCCACAAC ATCGAGGACGGCAGCGTGCA GCTCGCCGAC CACTACCAGC AGAACACCCCCATCXGGCGA CGGCCCCGTG CTGCTGCXXC CGACAACCAC SEQ ID NO: 7CAACGTCTAT ATCATGGCCG ACAAGCAGAA GAACGGCATCXXXAAGGTGA ACTTCAAGAT CCGCCACAAC ATCGAGGACGGCAGCGTGCA GCTCGCCGAC CACTACCAGC AGAACACCCCCATCXGGCGA CGGCCCCGTG CTGCTGCXXC CGACAACCAC SEQ ID NO: 8GCTGAAGCAC TGCACGCCGT AGGTCAGXXG GTGGTCACGAGGGTGGGCCA GGGCAXCGGG CAGCTTGCCG GTGGTGCAGATGAACTTCAG GGTCXXXAGC TTGCCGTAGG TGGCATCGXX XCCCTCGCCC TCGCCGSEQ ID NO: 9 GACACGCTGA ACTTGTGGCC GTTTACGTCG CCGTCCAGCTCGACCAGGAT GGGCXXXACC ACCCCGGTGA ACAGCTCCTCGCCCTTGCTC ACCXXXATGG TTGTGGCCAT ATTATCATCGTGTTTTTCAA AGGAAAACCA CGTCCCCGTG GTTCGGGGGGCCTAGACGTT TTTTTAACCT CGACTAAACA CATXGTAAAGCATGTGTACC GAGGCCCCAG ATCAGATCCC ATACA SEQ ID NO: 10AGGGCACGGG CAGCTTGCCG GTGGTGCAGA TGAACTTCAGGGTCAGCTTG CCGTAGGTGG CATCGCCCTC GCCCTCGCCXXXGGACACGC TGAACTTGTG XXXXXXGCCG TTTACGTCGCCGTCCXXXXX XXXAGCTXXX XXXXXXCGAC CAGGATGGGCACCACCCCGG TGAAXXCAGC TCCTCGCCCT TGCTCACCATXGGTXXXXXT GTGGCCATAT TATCATCGTG TTTXXXXXXXXXXTTCAAAG GXXXXAAAAC CACXGTCCCX CGTGGTTCGGGGGGCCTAGA CGTTTTTTTA ACCTCGACTA AAXXXXCACATGTAAAGCAT GTGTACCGAG GCXXXXXXXX XXXCCCAGATCAGATCCCAT ACAATGGGGT ACCTTCTGG

1. A method for improving cardiac function in a mammal, the methodcomprising: administering to the mammal a composition comprising aparticle encapsulating an mRNA, the mRNA encoding a polypeptide usefulfor regenerating cardiac function or regenerating tissue, therebyimproving cardiac function of the mammal.
 2. The method of claim 1,wherein the polypeptide is NAP-2, TGF-α, ErBb3, VEGF, IGF-1, FGF-2,PDGF, IL-2, CD19, CD20, or CD80/86.
 3. The method of claim 1, whereinthe mammal is a human.
 4. The method of claim 3, wherein the human hasundergone percutaneous coronary intervention for ST-elevation myocardialinfarction.
 5. The method of claim 1, wherein the composition isadministered to the mammal by arterial administration.
 6. The method ofclaim 1, wherein the particle comprises alginate.
 7. The method of claim6, wherein the alginate is in the form of an alginate gel.
 8. The methodof claim 7, wherein the alginate gel comprises a calcium salt.
 9. Themethod of claim 8, wherein the alginate gel has a ratio of alginate tocalcium salt can be from about 2:1 to about 10:1.
 10. The method ofclaim 1, wherein the particle is from about 5 μm to about 10 μm indiameter.
 11. The method of claim 1, wherein the particle is a biphasicparticle.
 12. The method of claim 11, wherein the biphasic particle is apolarized particle.
 13. The method of claim 11, wherein the biphasicparticle comprises a tail.
 14. The method of claim 1, wherein the methodcomprises administering the composition during a percutaneous coronaryintervention.
 15. The method of claim 1, where the particle furthercomprises a scaffold protein.
 16. The method of claim 15, where thescaffold protein is selected from the group consisting of collagen I,collagen II, collagen III, collagen IV, fibrin, and gelatin.
 17. Themethod of claim 1, where the particle further encapsulates apolypeptide.
 18. The method of claim 17, where the polypeptide is anantibody having the ability to neutralize tumor necrosis factoractivity, an antibody having the ability to neutralize mitochondrialcomplex-1 activity, or a resolvin-D1 agonist.
 19. The method of claim 1,where the particle further encapsulates a lipopolysaccharide.
 20. Themethod of claim 1, wherein the particle further encapsulates amicrovesicle and/or exosome.
 21. A method for improving cardiac functionin a mammal, the method comprising: administering to the mammalcomposition comprising a particle encapsulating an inhibitory RNA, theinhibitory RNA reducing expression of a polypeptide in which reducedexpression of the polypeptide improves cardiac function, therebyimproving cardiac function of the mammal, wherein the polypeptide iseotaxin-3, cathepsin-S, DK-1, follistatin, ST-2, GRO-a, IL-21, NOV,transferrin, TIMP-2, TNFaRI, TNFaRII, angiostatin, CCL25, ANGPTL4, orMMP-3.
 22. The method of claim 21, wherein the mammal is a human. 23.The method of claim 22, wherein the human has undergone percutaneouscoronary intervention for ST-elevation myocardial infarction.
 24. Themethod of claim 21, wherein the composition is administered to themammal by arterial administration.
 25. The method of claim 21, whereinthe particle comprises alginate.
 26. The method of claim 25, wherein thealginate is in the form of an alginate gel.
 27. The method of claim 26,wherein the alginate gel comprises a calcium salt.
 28. The method ofclaim 27, wherein the alginate gel has a ratio of alginate to calciumsalt can be from about 2:1 to about 10:1.
 29. The method of claim 21,wherein the particle is from about 5 μm to about 10 μm in diameter. 30.The method of claim 21, wherein the particle is a biphasic particle. 31.The method of claim 30, wherein the biphasic particle is a polarizedparticle.
 32. The method of claim 30, wherein the biphasic particlecomprises a tail.
 33. The method of claim 21, wherein the methodcomprises administering the composition during a percutaneous coronaryintervention.
 34. The method of claim 21, where the particle furthercomprises a scaffold protein.
 35. The method of claim 34, where thescaffold protein is selected from the group consisting of collagen I,collagen II, collagen III, collagen IV, fibrin, and gelatin.
 36. Themethod of claim 21, where the particle further encapsulates apolypeptide.
 37. The method of claim 36, where the polypeptide isselected from the group consisting of an antibody having the ability toneutralize tumor necrosis factor activity, an antibody having theability to neutralize mitochondrial complex-1 activity, and aresolvin-D1 agonist.
 38. The method of claim 21, where the particlefurther encapsulates a lipopolysaccharide.
 39. The method of claim 21,wherein the particle further encapsulates a microvesicle and/or exosome.40. A composition comprising: a particulate substrate; an mRNA attachedto the particulate substrate, the mRNA comprising at least onemodification to inhibit degradation of the mRNA when the mRNA is incytosol of a cell, the mRNA encoding at least one therapeuticpolypeptide.
 41. The composition of claim 40, wherein the mRNAmodification comprises a pseudoknot, an RNA stability element, or anartificial 3′ stem loop.
 42. The composition of claim 40, wherein theparticulate substrate comprises a surface modification.
 43. Thecomposition of claim 42, wherein the surface modification comprises abiocompatible polymer.
 44. The composition of claim 43, wherein thebiocompatible polymer comprises polyethylene glycol (PEG).
 45. Thecomposition of claim 44, wherein the biocompatible polymer compriseschitosan.
 46. The composition of claim 40, wherein the particulatesubstrate comprises a nanoparticle.
 47. The composition of claim 40,wherein the particulate substrate comprises a microparticle.
 48. Thecomposition of claim 40, wherein the particulate substrate comprises aplurality of nanoparticles.
 49. The composition of claim 48, wherein theplurality of nanoparticles comprises: at least one nanoparticlecomprising from a first material; and at least one nanoparticlecomprising a second material.
 50. The composition of claim 40, whereinthe therapeutic polypeptide comprises: an immunoglobulin heavy chain; oran immunoglobulin light chain.